Viral immune evasins impact antigen presentation by allele-specific trapping of MHC I at the peptide-loading complex

Major histocompatibility complex class I (MHC I) molecules present antigenic peptides to cytotoxic T cells to eliminate infected or cancerous cells. The transporter associated with antigen processing (TAP) shuttles proteasomally generated peptides into the ER for MHC I loading. As central part of the peptide-loading complex (PLC), TAP is targeted by viral factors, which inhibit peptide supply and thereby impact MHC I-mediated immune responses. However, it is still poorly understood how antigen presentation via different MHC I allotypes is affected by TAP inhibition. Here, we show that conditional expression of herpes simplex viral ICP47 suppresses surface presentation of HLA-A and HLA-C, but not of HLA-B, while the human cytomegaloviral US6 reduces surface levels of all MHC I allotypes. This marked difference in HLA-B antigen presentation is echoed by an enrichment of HLA-B allomorphs at US6-arrested PLC in comparison to ICP47-PLC. Although both viral factors prevent TAP-mediated peptide supply, our data imply that MHC I allomorphs favor different conformationally arrested states of the PLC, leading to differential downregulation of MHC I surface presentation. These findings will help understand MHC I biology in general and will even advance the targeted treatment of infections depending on patients’ allotypes.

www.nature.com/scientificreports/ independently of each other acquired highly efficient ways to block TAP-mediated peptide transport 23 . Upon TAP inhibition, empty MHC I molecules are retained in the ER lumen and are consequently unable to display antigens at the cell surface to the immune system 18,19,[23][24][25][26][27][28] . Remarkably, the viral inhibitors identified so far lack structural homology, bind to different regions, and arrest distinct conformations of the TAP complex 17,19,29 . Common to all TAP inhibitors is that they arrest the transporter in a transport-incompetent state, thereby blocking peptide supply to the ER lumen. This shortage of peptides, in turn, leads to a reduction of peptide-loaded MHC I molecules on the cell surface and helps the virus escape the host immune response 17,20,21,30 . The immediate early ICP47 of Herpes simplex virus (HSV1) blocks translocation of peptides by wedging into the peptide-binding cleft of the TAP transporter without affecting ATP binding [31][32][33][34][35] . In contrast, the unique short sequence 6 (US6) encoded by human cytomegalovirus (HCMV) is an ER-resident type-I transmembrane glycoprotein with an active ER-lumenal domain interacting with human TAP, which inhibits TAP by a distinct mechanism from ICP47 [36][37][38][39][40][41][42] . Infection with HSV2, particularly in the presence of ICP47, was shown to specifically down-regulate HLA-C surface presentation in human lymphoblastoid cell lines 13 . US6 was shown not to inhibit cell-surface expression of TAP-independent HLA-A*02:01 but to reduce the cell-surface expression of HLA-B*27:05 (ref. 43 ). However, the mechanism by which such viral factors affect the different MHC I allomorphs has not been addressed so far. We therefore conducted a comprehensive analysis of the cellular and biochemical consequences of ICP47 and US6 expression on PLC composition. Expression of the immune evasins during viral infection was mimicked by a tightly regulated conditional transcription of the viral genes in different stably transduced cell lines. We uncovered a differential cell surface presentation of MHC I allomorphs upon TAP inhibition by ICP47 or US6, which were consistent with the alterations in the MHC I composition in the PLC arrested by ICP47 or US6. Our results reveal a molecular mechanism behind allele-specific blockage of MHC I antigen presentation and provide opportunities for the development of new treatments for infectious diseases.

Results
Stable cell lines for the conditional expression of ICP47 or US6. To compare the effects of the immune evasion factors on MHC I surface presentation, we generated stably transduced Raji and Mel JuSo cell lines for conditional expression of ICP47 or US6 using a doxycycline-inducible expression system 44 . Both viral inhibitors were equipped with a C-terminal SBP-tag for affinity purification, followed by an internal ribosomal entry site (IRES2) driving bicistronic expression of mCherry as a fluorescent reporter. Upon induction with doxycycline, expression of the viral factors was monitored by flow cytometry using mCherry and by immunoblotting of the cell lysates against the SBP-tag of each viral factor (Fig. 1). Upon doxycycline induction, bicistronic expression of mCherry was observed in > 65% of Raji cells (Fig. 1a,b) and > 75% of Mel JuSo cells (Supplemen- Figure 1. Conditional expression of ICP47 and US6 in Raji cells. (a) Raji cells were stably transduced with ICP47-or US6-encoding lentivirus. The expression of ICP47 and US6 was induced with 2 µg ml −1 doxycycline (Dox) for 16 h. Protein expression was monitored by bicystronic mCherry expression via flow cytometry. The histogram shows mCherry expression in ICP47-(orange/red) and US6-encoding (cyan/blue) Raji cells and wild-type Raji cells (grey/black) upon doxycycline induction. (b) Quantification of mCherry expression in ICP47 (orange/red bar) and US6 (cyan/blue bar) expressing cells in comparison to the non-induced (grey/black bar) cells (n = 2, mean ± SD). (c) Immunoblot analysis of TAP1, HLA-A, HLA-B, HLA-C, and SBP-tag in total cell lysates of stably transduced non-induced or induced Raji cells (ICP47 or US6 transduced) or wild-type Raji cells (-). GAPDH was used as a loading control. The quantification of densitometries of TAP1, HLA-A, HLA-B, and HLA-C normalized to the loading control GAPDH in the whole cell lysate of doxycycline-induced cells compared to non-induced cells is depicted below the immunoblots. www.nature.com/scientificreports/ tary Fig. 1a,b) in comparison to the non-induced cells. To establish homogenous cell populations, we generated monoclonal cell lines by subjecting the transduced Raji or Mel JuSo cells to fluorescence-activated cell sorting (FACS) gated on doxycycline-induced mCherry expression ( Supplementary Fig. 1d). To assess the overall effects of the conditional expression of the viral factors, we compared the protein expression levels of TAP1, HLA-A, HLA-B, and HLA-C in cell lysates derived from non-induced and induced cells by quantitative immunoblotting (Fig. 1c). Upon doxycycline induction, we did not observe a substantial variation in the overall expression of TAP1 and different MHC I allomorphs in either Raji (HLA-A*03:01, HLA-B*15:10, and HLA-C*04:01) (Fig. 1c) or Mel JuSo cells (HLA-A*01:01, HLA-B*08:01, and HLA-Cw7) ( Supplementary Fig. 1c), demonstrating that the protein expression levels of central components of the antigen presentation pathway are not altered upon expression of ICP47 or US6. Thus, specific changes in the steady-state levels of the MHC I allomorphs by synthesis and degradation can be excluded.

Induced expression of ICP47 or US6 blocks TAP-mediated peptide supply into the ER. To
show that induced expression of ICP47 and US6 blocks peptide translocation into the ER lumen, we performed single-cell-based transport assays 45 . This approach relies on the TAP-dependent transport of fluorescently labeled reporter peptides in semi-permeabilized cells and their subsequent accumulation in the ER lumen [45][46][47] . We demonstrate that ATP-dependent transport of the RRYQNSTC AlexaFluor647 L peptide (NST AF647 ) is inhibited in doxycycline-induced cells expressing ICP47 and US6, while the parental and non-induced cells displayed a high level of ATP-stimulated transport (Fig. 2a,b). These data demonstrate that the induced expression of ICP47 or US6 in the monoclonal cell lines prevents TAP-mediated peptide supply into the ER.  Table 1). As a gold-standard for antibody evaluation, we www.nature.com/scientificreports/ transfected HAP1 HLA-A/B/C knockout (HLA KO ) cells 48 with plasmids encoding each HLA allomorph and subsequently monitored the expression by immunostaining and flow cytometry ( Supplementary Fig. 3). We observed no cross-reactivity of the allomorph-specific antibodies, neither in immunoblotting nor flow cytometry ( Supplementary Fig. 3). In Raji cells, we found a > 95% reduction in surface expression of the HLA-A and HLA-C allomorphs in comparison to non-induced cells (Fig. 3a,b). Similarly, in Mel JuSo cells expressing HLA-A*01:01, HLA-B*08:01, and HLA-Cw7, the HLA-A and HLA-C surface presentation was reduced by ~ 80% and > 90%, respectively, as compared to non-induced cells ( Supplementary Fig. 2a,b). Surprisingly, surface presentation of HLA-B was differentially affected by both viral factors. While US6-expressing Raji cells showed a drastic reduction of HLA-B*15:10 surface presentation (> 70%), its surface presentation was hardly affected upon expression of ICP47 ( Fig. 3a,b). In Mel JuSo cells, the impact on HLA-B surface presentation was even further amplified, leading to a threefold increase of HLA-B*08:01 upon expression of ICP47 when compared to a 99% reduction upon expression of US6 ( Supplementary Fig. 2a,b). It is worth mentioning that doxycycline addition did not change MHC I cell surface presentation, thus excluding indirect or unspecific effects (Fig. 3a,b and Supplementary Fig. 2a,b). Overall, these findings indicate that although expression of viral TAP inhibitors causes an overall reduction of MHC I cell surface presentation by preventing peptide supply, their effects on the individual MHC I allomorphs, in particular HLA-B allomorphs, are highly variable.

Viral factors differentially impact the MHC I composition of the PLC. Raji cells display high
expression levels of PLC, which provided us with a key advantage to analyze the assembly of all PLC components in response to viral factor expression. Moreover, Raji cells can be grown in suspension to high density allowing purification of native PLCs in high quality and quantity 49 . To elucidate the effect of ICP47 and US6 expression on the individual MHC I allomorphs, we isolated native ICP47-or US6-arrested PLCs from glyco-diosgenin (GDN)-solubilized Raji cell lysates using the SBP-tag on ICP47 or US6 (Fig. 4). The PLCs were isolated as welldefined monodisperse macromolecular complexes. We then compared the MHC I composition of ICP47-and US6-arrested PLC to that of native PLC, which was isolated via the anti-TAP1 antibody (mAb148.3) and eluted with the specific epitope PADAPE 50 . Native PLCs were subsequently purified by size exclusion chromatography (SEC), and peak fractions were analyzed by SDS-PAGE (Fig. 4a) and immunoblotting against the three different MHC I allomorphs (Fig. 4b). Densitometric analyses revealed a three-fold enrichment of HLA-B in US6arrested PLC compared to ICP47-PLCs, while the amounts of co-precipitated HLA-A and HLA-C allotypes remained comparable in all purified PLCs (Fig. 4c).
To further corroborate the differential effects of viral factors on the MHC I composition in ICP47-and US6arrested PLCs, stoichiometries in monodisperse complexes were analyzed by label-free quantification mass spectrometry (MS) (Fig. 5a). For this, the purified PLC complexes (Fig. 5b) were hydrolyzed with trypsin, and www.nature.com/scientificreports/ the obtained peptides were analyzed by liquid chromatography-coupled tandem-MS (LC-MS/MS). Protein intensities were then obtained by label-free quantification using MaxQuant 51,52 . Subsequently, intensity-based absolute quantification (iBAQ) 53 was used for relative comparison of all PLC components. For this, peptide intensities of each protein were summed and normalized by the number of theoretically observable peptides. PLC components were then normalized in relation to TAP1 in ICP47-PLC, showing a fully assembled PLC (Fig. 5c). iBAQ values of the MHC I allomorphs in ICP47-and US6-arrested PLC confirmed a three-fold enrichment of HLA-B in the US6-arrested PLC compared to the ICP47-inhibited PLC (Fig. 5d). Like the immunoblot analysis, ratios of HLA-A and HLA-C allotypes remained unchanged when ICP47-and US6-arrested PLCs were compared (Fig. 5d). These findings demonstrate that, though both viral factors block TAP and prevent ER peptide supply, ICP47 and US6 have a very different impact on the PLC regarding the recruitment of HLA-B allomorphs.

Discussion.
In this study, we demonstrated that the viral inhibitors ICP47 and US6 alter the MHC I composition of the PLC, thus providing the molecular basis for the different impact on the cell surface expression of specific MHC I allomorphs. To study the effect of viral TAP inhibitors in the absence of potential overlapping factors, we generated monoclonal cell lines, which conditionally express ICP47 or US6 (Fig. 1). Upon doxycycline induction, the expression of ICP47 or US6 completely abrogated TAP-dependent peptide transport (Fig. 2) and led to an overall down-regulation of the MHC I surface presentation (Fig. 3). To our surprise, ICP47 and US6 had different effects on the surface expression of each HLA allotype: in comparison to ICP47 expression, US6 expression caused a three-fold stronger inhibition of HLA-B cell-surface levels (Fig. 3). However, a similarly reduced surface level of both, HLA-C (> 98% inhibition compared to the wild type), and HLA-A (> 96% inhibition compared to the wild type) was observed upon ICP47 and US6 expression (Fig. 3).
Since no global effect on the MHC I expression level was observed in cell lysates in which ICP47-and US6expressing cells were compared with non-induced cells (Fig. 1c), we rationalized that preferential trapping of MHC I in the ICP47-or US6-arrested PLC was the reason for the different surface levels of HLA allomorphs. As analyzed by co-immunoprecipitation followed by immunoblotting and mass spectrometry, the MHC I repertoire of purified ICP47-PLC and US6-PLC showed a three-fold enrichment of HLA-B in US6-arrested PLC compared to ICP47-arrested PLC. There is no evidence to date that suggests a direct interaction between the TAP inhibitors and MHC I in the PLC. However, such distinct allomorph-specific trapping at the PLC together with a differential reduction in cell surface presentation is consistent with the missing-self hypothesis, which proposes that cells lacking MHC I molecules on their cell surface are targeted by natural killer (NK) cells. HCMV encodes the MHC I mimic UL18 to hide infected cells from NK cells; however, no such molecule has been identified in HSV. Therefore, we argue that HSV needs to allow presentation of HLA-B allomorphs to escape recognition by NK were normalized to TAP1 (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3, mean ± SD). Statistical analysis was performed using two-way ANOVA. ns: non-significant. www.nature.com/scientificreports/ cells. Moreover, the HLA-B allomorphs presented on the cell surface of ICP47-expressing cells likely display an altered peptide repertoire of host origin to evade NK cell recognition -and additionally-inhibit the activation of a CD8 + T cell response. ICP47 and US6 are structurally different and vary in their mode of inhibition 35,36,40,49 . ICP47, a soluble protein with a helix-loop-helix structure 54 , targets TAP from the cytosol and competes with peptide binding 31,42 . The active, ER-lumenal domain of US6 allosterically arrests TAP interfering with ATP binding on the cytosolic side of the ER membrane 39,40 . Thus, the inhibition mechanisms of ICP47 and US6 are fundamentally different and these viral factors arrest the TAP complex in different conformations 41,42 . Our data suggest that the different conformational states of the arrested TAP complex results in the altered MHC I allomorph composition in the PLC and is independent of abated peptide supply to the ER. Therefore, we hypothesize that the conformational difference between ICP47-versus US6-arrested TAP results in the preferential recruitment of HLA-B in US6-arrested PLCs compared to ICP47-arrested PLCs. A high-resolution structure of US6arrested PLC will help understand the detailed mechanism of MHC I allomorph restriction. The present study also indicates that different viral immune evasins have evolved strategies to selectively down-regulate specific HLA allotypes that are actively involved in the surface presentation of viral peptides to CTLs to evade the host immune response. Although we could show the same effect in two unrelated cell lines, a lymphoma (Raji) and a melanoma cell line (Mel JuSo), larger studies need to be conducted to show a general effect with the many different HLA-B allomorphs known to date. Like ICP47 and US6, other virally encoded immune evasins, such as Epstein-Barr virus BNLF2a, cowpox virus CPXV12, and bovine herpes virus UL49.5, vary greatly in terms of structure and topology and have different modes of TAP inhibition [55][56][57][58][59][60] . Whether down-regulation of MHC I surface expression by these viral inhibitors results from a differential impact on the MHC I repertoire at the PLC needs to be addressed in future studies. In addition, how the down-regulation of specific HLA allomorphs by viral immune evasins modulate the host T-cell response remains an open question. www.nature.com/scientificreports/
Cell lines. All cell lines used in the present study were cultured at 37 °C in a humidified atmosphere with 5%  www.nature.com/scientificreports/ Isolation of human PLCs. The harvested cells were thawed on ice and resuspended in solubilization buffer: 20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 1% (w/v) protease inhibitor mix, and 2% (w/v) GDN. 5 ml of the solubilization buffer was added per gram of cells. The lysate was homogenized and incubated at 4 °C for 1 h. The lysate was centrifuged at 100,000 ×g for 1 h at 4 °C, and the cleared supernatant was incubated with pre-equilibrated streptavidin agarose beads (ThermoFisher, Pierce). After washing the beads three times with washing buffer (20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 0.02% (w/v) GDN, and 2 mM PMSF), the protein was eluted with 2.5 µM biotin in 20 mM HEPES-NaOH pH 7.4, 150 mM NaCl, and 0.02% (w/v) GDN. The eluate was concentrated with a 100-kDa cut-off concentrator (Amicon-Ultra 0.5 ml, Merck, Millipore). The PLCs were isolated by size exclusion chromatography (Superose 6 3.2/300, GE), and the peak fraction harvested for further analysis.
Peptide transport assay. 0.2 × 10 6 Raji cells were semi-permeabilized using streptolysin O (2 U ml −1 ) at 4 °C for 15 min and washed to remove residual SLO. Transport was carried out in the presence of 10 mM ATP or ADP, 10 nM NST A647 in PBS buffer supplemented with 10 mM MgCl 2 for 20 min at 37 °C in a 50 µl reaction volume. The transport was stopped by addition of 150 µl PBS supplemented with 20 mM EDTA. The peptide transport was monitored by flow cytometry, and the data were analyzed using FlowJo (TreeStar) software reporting the mean fluorescence intensity.
Immunoblotting. Protein samples were heated to 95 °C for 10 min with a reducing SDS-sample loading buffer. The samples were analyzed by SDS-PAGE (12%) and subsequent electroblotting onto methanol-activated polyvinylidene fluoride (PVDF) membranes. The blotting membrane was blocked with non-fat milk before incubating with the primary antibody against human TAP1 (mAB148.3), HLA-A/B/C (W6/32), HLA-A (Ab52922), HLA-B (Ab76795), and HLA-C (Ab126722). The immunoblots were analyzed by chemiluminescence using the corresponding secondary antibody and the Clarity Western ECL reagent (BioRad). Chemiluminescence was recorded with a Lumi-Imager (Roche). The signals of the immunoblots were quantified using ImageJ (NIH).
Flow cytometry. The induction of protein production by doxycycline in stably transduced cells was monitored by flow cytometry (FACS melody, BD Biosciences) using mCherry as reporter. MHC I surface expression of the induced and non-induced cells was monitored with monoclonal antibodies listed in Supplementary  Table 1. For surface staining, cells were harvested 14-16 h after induction. Cells were washed twice in ice-cold FACS buffer (1 × DPBS, 2 mM EDTA, 1% BSA). Antibody surface staining procedures were carried out on ice. Cells were first incubated with an FcR-receptor blocking reagent (BioLegend). For direct antibody staining, cells were incubated with an APC-conjugated primary antibody for 30 min in the dark. After washing twice, cells were resuspended in FACS buffer and analyzed by flow cytometry. For indirect staining, cells were stained with an unconjugated primary antibody, washed, and incubated with corresponding APC-conjugated secondary antibody for 20 min. Cells were washed twice and MHC I surface expression was monitored by flow cytometry, and the data were analyzed using FlowJo (TreeStar). The median fluorescent intensities (MFI) were calculated and compared.
LC-MS/MS sample preparation. 50 µg of PLC proteins were precipitated using 1/10 vol. 3 M sodium acetate, pH 5.3 and 3 vol. ice-cold ethanol followed by overnight incubation at − 20 °C. The protein pellet was washed twice with 80% (v/v) ice-cold ethanol and then dried in a vacuum centrifuge. Tryptic digestion in the presence of RapiGest (Waters) was performed according to the manufacturer's protocol. Briefly, the protein pellet was dissolved in 10 µl 1% (w/v) RapiGest in 25 mM ammonium bicarbonate, pH 8.5, and incubated for 15 min at room temperature. Cysteines were reduced by addition of 10 µl of 50 mM dithiothreitol in 25 mM ammonium bicarbonate, pH 8.5, and by incubation for 30 min at 60 °C. Cysteines were alkylated with 10 µl 100 mM 2-iodoacetamide in 25 mM ammonium bicarbonate, pH 8.5, for 30 min at 37 °C in the dark. Prior to tryptic hydrolysis, the RapiGest concentration was diluted to 0.1% (w/v) with 25 mM ammonium bicarbonate, pH 8.5. For protein digestion, trypsin (Promega) was added at an enzyme-to-protein ratio of 1:20 (w/w) followed by incubation at 37 °C overnight. Subsequently, the RapiGest was degraded by addition of 20 µl of 5% (v/v) trifluoroacetic acid and incubation for 2 h at 37 °C. Degraded RapiGest was removed by centrifugation, and peptides were dried in a vacuum centrifuge.